The cellular origins of helper T cell memory and the regulation of its development in vivo have been the focus of intense research in recent years. Unraveling the mechanisms controlling effective memory formation is of fundamental interest with potentially high impact on the public health initiative of vaccine design. Insights into the complex cellular processes that underlie the development of Th cell immunity may also reveal new treatment modalities for a broad range of clinically-relevant conditions such as autoimmune disease, transplant rejection, and various infectious diseases. Immunological memory is a multi-faceted process whose main purpose is long-term protection precisely targeted to previously- experienced antigens. Th cell memory is a systemic phenomenon characterized by the rapid emergence of effector Th cells in response to rechallenge with antigen. My laboratory is primarily interested in the cellular origins of this rapid memory response to antigen and the regulation of its development in vivo. We hypothesize that Th cell memory is organized in multiple cellular compartments, each sub-specialized to regulate long-term antigen- specific protection. We use a murine model of protective immunity to a non-replicating protein antigen to focus Th cell activity on the development of antigen-specific B cell immunity. We have developed a flow cytometric strategy to quantify, phenotype, and isolate antigen-specific Th cells at various stages after initial priming and antigen rechallenge in vivo. We propose to study clonal selection, function, and regulation of antigen-specific Th cell immunity directly ex vivo, post-adoptive transfer in vivo and using single cell analyses of function in vitro. The seminal work of Kupfer, Dustin, and others have identified a molecular process that serves to organize cognate inter-cellular interactions that are now commonly referred to as the formation of an immunological synapse. During the development of an immune response, antigen-specific Th cells will form immune synapses with a variety of different cell types, often in dynamic microenvironments specifically associated with the response to antigen. Here, we outline a Serial Synapsis Model for the ordered development of antigen-specific Th cell immunity. This model integrates our understanding of the progression of cellular events in vivo with overlapping phases of synapse dependent and synapse independent lymphocyte development. Using this model as a framework, we will pursue our central hypothesis in three broad specific aims. (1) To understand the mechanisms that underlie clonal selection in the Th cell compartment during an immune response and assess what contribution the pre-immune repertoire plays in the development of clonal dominance. (2) To evaluate quantitatively the spectrum of Th cell function that develops as a consequence of antigen experience. (3) To probe the changes in Th cell physiology that accompany developmental progression from naive to effector to memory Th cells in vivo.